The agglutination reaction has long been used in visual (semi-quantitative) and quantitative assays for a wide variety of bacteria, cell-surface antigens, serum proteins and other analytes of clinical interest. Agglutination results from the reaction of bivalent antibodies with multivalent antigens of interest to produce aggregates which can be detected and/or measured in various ways. Similarly, the same reaction can be utilized for the detection of specific antibodies by the agglutination reaction caused by the addition of the corresponding antigen.
Generally, in order to produce large, cross-linked aggregates, the number of reactive sites on the antigens must be at least 2. When the detection of monovalent haptens is desired, the reaction scheme has to be modified as follows: a multivalent form of the antigen such as a hapten-protein conjugate is prepared and the free hapten present in a sample competes with its multivalent form for the available binding sites of the antibody, thereby reducing the amount of agglutination. This technique is referred to as inhibition of agglutination.
Production of multivalent forms of haptens is old in the art. Frequently, the hapten is bonded to a carrier protein as is done in the preparation of immunogens. The stoichiometry of the reaction can be adjusted to place three or more haptens per protein molecule, the exact number determined by the needs of the particular assay in which the material will be utilized.
Increased sensitivity to visual or instrumental detection of agglutination or its inhibition can be achieved by the use of particle reagents as carriers, rather than soluble proteins or protein conjugates. It has been shown, for example, that antiserum to hen ovalbumin was 2000-fold more sensitive in precipitating hen ovalbumin coated on colloidion particles than in precipitating hen albumin itself; H. N. Eisen, "Immunology", Harper and Row, 1974, page 394.
Antibody particle reagents are also known. A common method for preparation of such reagents is the adsorption of the antibodies onto the surface of suitable adsorbents. Polystyrene-based latex particles have been used extensively for this purpose. These reagents, however, are susceptible to desorption during storage or use leading to variations in reagent properties. This, in turn, can adversely affect assay sensitivity and reproducibility.
To overcome the problems of desorption, particle reagents can be prepared by covalent attachment of the compounds of biological interest to the surface of particles. Polystyrene polymers have been modified to include functional groups capable of covalent protein attachment. U.S. Pat. No. 4,064,080, issued Dec. 20, 1977, discloses styrene polymers with terminal aminophenyl groups and proteins attached to them. U.S. Pat. No. 4,181,636, issued Jan. 1, 1980, discloses carboxylated latex polymers coupled to immunologically active materials through a water soluble activating agent and their use as diagnostic reagents in agglutination tests. U.S. Pat. No. 4,210,723, issued July 1, 1980, describes shell-core latex polymer particles of 0.15-1.5 .mu.m diameter having free epoxy groups on the surface of the particles and the coupling of proteins through these epoxy groups.
Other polymeric systems have also been developed for later attachment of immunologically active materials. U.S. Pat. No. 4,264,766, issued Apr. 28, 1981, discloses latex polymers having a particle size of 0.01-0.9 .mu.m and having active groups such as carboxyl and amino groups to which water soluble polyhydroxy compounds can be attached covalently. Through the utilization of activating agents such as carbodiimides, immunologically active materials were attached to the latex particle/polyhydroxy compound carriers to form diagnostically useful reagents.
U.S. Pat. No. 4,401,765, issued Aug. 30, 1983 to Craig et al., discloses shell-core polymer particles wherein the inner core has a high refractive index resulting in high sensitivity to light scattering measurements and the outer shell contains functional groups to which compounds of biological interest can be attached directly or through a proteinaceous material
Usually, low molecular weight compounds of biological interest (analytes) are not attached directly to latex particles. The reason for this is that such particle reagents often do not aggregate well when mixed with the appropriate antibody or that they lack functional groups appropriate for covalent attachment to the particle surface. To overcome these problems analytes are usually linked to particles via a bridge or spacer moiety.
The most commonly used spacers are proteins or glycoproteins, such as albumin. Human serum albumin (HSA) is a water-soluble protein having an abundance of amino groups which can be utilized in coupling to amino-reactive groups both on the analyte and on the particle surface.
Although particle reagents prepared from analyte-protein conjugates have shown great utility in general, there are still some disadvantages associated with their use. The solubility and stability characteristics of proteins limit the chemical means which can be used in forming the analyte-protein conjugate and attaching it to the particle. Proteins are denatured by extremes of temperature and pH and are frequently insoluble in organic solvents. Furthermore, even hydrophilic proteins have hydrophobic regions in which hydrophobic analytes can be buried. Extended storage of the particle reagent can result in protein hydrolysis, oxidation, conformational changes, and microbial degradation. Finally, the protein spacer can contain antigenic determinants which are recognized by antianalyte antibodies, resulting in nonspecific agglutination during an assay.
There is a need for hydrophilic, chemically well-defined linking groups which can be utilized in the preparation of particle reagents for use in clinical diagnostics.